Glioblastoma multiforme (GBM) has an abysmal prognosis despite advances in genomics, new targeted therapies, and increased understanding of its pathobiology. The goal of our Program Project Grant (PPG) is to improve knowledge about GBM biology and develop effective therapies. Investigators in this PPG have made two notable advances, among many: 1- Tumor extracellular vesicles (tEVs) and their contents transfer functional information among cells in the GBM microenvironment, thereby increasing oncogenic behaviors, and 2- a Phase II clinical trial in patients with newly diagnosed GBM utilizing Gene-mediated Cytotoxic Immunotherapy (GMCI) as an adjuvant to standard-of-care (SOC) has shown encouraging, albeit non- definitive, results. The major hypotheses of this PPG's competitive renewal are that GBM therapy can be improved by combining SOC and GMCI with immune checkpoint inhibition, and that therapeutic responses can be monitored in biofluids by assessing protein and RNA content in tEVs. Three synergistic projects and three supporting cores will test these hypotheses. Project 1 (Xandra Breakefield) will evaluate whether glioma EVs and their contents are fundamental regulators of glioma heterogeneity within the tumor and immune suppression in the tumor microenvironment, thus contributing to tumor progression, immuno-evasion, and therapeutic resistance. Project 2 (Ralph Weissleder) will validate novel technological advances from his laboratory, such as single EV analysis (SEA), to assay tEV contents in biofluids, thus overcoming the limitation of scant materials available from preclinical and clinical trials. Project 3 (E. Antonio Chiocca) will test if SOC/GMCI combined with immune checkpoint inhibition will be an effective therapy in preclinical models of glioma and, ultimately, in a randomized clinical trial. Core A (Breakefield/Chiocca) will provide the necessary administrative structure, scientific oversight, and overall leadership functions for the PPG, including basic science (Breakefield) and preclinical/clinical (Chiocca) efforts. Core B (Carter) will maintain the Clinical Sample Core that biobanks cerebral spinal fluid and serum/plasma samples from patients and clinical trials for tEV analysis, as well as serve as the biostatistics resource. Core C (Charest) will provide genetically engineered mouse models of gliomas and mouse/human glioma ?stem-like? cells from different GBM subtypes. Together these investigators will generate the necessary scientific justification and preclinical data to ultimately support the proposed clinical trial of GMCI with immune checkpoint inhibition in newly diagnosed GBM patients, in which tEVs will be evaluated for their potential as biomarkers and modulators of the therapeutic response.